Method of using low molecular weight hyaluronic acid for stimulating bone formation

ABSTRACT

The present invention is drawn to hyaluronic acid fractions having molecular weights in the range of from about 20,000 to about 60,000 daltons, wherein these hyaluronic acid fractions possess osteoinductive activity. The present invention further includes pharmaceutical compositions comprising such hyaluronic acid fractions to be used in the treatment of various types of bone disorders, as well as methods of treating such disorders using the pharmaceutical compositions.

This application is the U.S. national stage entry under 35 U.S.C. 371 ofPCT/EP93/00932, filed on Apr. 16, 1993.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of various fractions ofhyaluronic acid as osteoinductive agents, i.e., agents capable ofstimulating the growth and differentiation of bone forming cells, andtherefore the formation of new bone material itself. The type ofactivity refers to cells of mammalian origin.

2. Description of Related Art

An intricate network of macromolecules constituting the extracellularmatrix, a substantial part of the tissue volume, largely fills theextracellular space. In many tissues, such as connective tissues, it isgenerally more abundant and completely surrounds the cells on all sides,thus determining the physical properties of the tissue. Theextracellular matrix is of considerable importance in regulating manyprocesses of tissue behavior. It has been demonstrated that changes inthe state and composition oft he extracellular matrix profoundlyinfluence biosynthetic processes and tissue development.

Glycosaminoglycans (GAGs) are the most plentiful non-fibrous,extracellular macromolecules, and are ubiquitous in all connectivetissues. This group of high molecular weight anionic glycoconjugates ispresent in both the soft and mineralized connective tissues. They aresubstantially composed of large molecules called "non-collagenousproteins" (NCP) of the extracellular matrix, or "proteoglycans". Thefirst of these terms distinguishes these molecules from the group offibrous proteins such as collagen, elastin, fibronectin and laminin; thesecond indicates that they are usually found to be covalently bound toprotein.

The biological properties of proteoglycans have been the object ofintense research, mainly with regard to the soft tissues. The bone andcement, on the other hand, have been virtually ignored as far as theirproteoglycan content is concerned. In fact, there is only one report inexistence which describes the proteoglycans of the alveolar bone (R. J.Waddington et al., Connective Tissue Res., 1988: 17, 171).

GAGs are long, unbranched polysaccharide chains composed of repetitivedisaccharide units. The type of sugar residues, their bonds, and thenumber and position of the sulfate groups have led to the identificationof four main groups of GAGs: 1) hyaluronic acid, 2) chondroitin solfateand dermatan sulfate, 3) heparan sulfate and heparin, and 4) keratansulfate. It is important to note that GAGs (with the probable exceptionof hyaluronic acid) rarely exist in a free state within tissues. Theyare usually covalently bound to proteins.

With the exception of hyaluronic acid, GAGs contain sulfate groups,which together with carboxy groups, form a molecule with a highlynegative charge under physiological conditions.

Hyaluronic acid (HA), also called hyaluronan or hyaluronate, contains upto several thousand sugar residues. It is a relatively simple moleculecomposed of regular sequences of non-sulfated disaccharide units.

HA is considered capable of facilitating cell migration duringmorphogenesis and tissue repair. It is found in varying quantities inall tissues and fluids in adult animals, and is particularly abundant inearly embryos. Because of its simplicity, HA could represent theearliest evolutionary form of glycosaminoglycan. There is a correlationbetween HA production and mesenchymal cell movement on the one hand, andbetween HA distribution and cell differentiation on the other (B. Tooleet al., Proc. Nat. Acad. Sci., USA, 1972: 69, 1384). The wound healingprocess possesses some aspects in common with early events which occurduring the embryonic development of many organs, and HA plays animportant role in both processes (B. P. Toole (1976) in S. H. Barondes,Ed., Neuronal Recognition, Plenum Press, New York, pp. 275-329); acorrelation has been found between HA and cell adhesion, in terms of HAreceptors (Goldstein et al., Cell, 1989: 56, 1063; I. Stamenkovic et al,Cell, 1989: 56, 1057).

Most of the characteristics of HA noted above for connective tissue canalso be observed in bone. The probability that noncollagenous proteinsinfluence local calcification mechanisms was assessed some time ago (H.Iwata et al., Clin. Orthop., Rel. Res. 1973: 90, 236; M. R. Urist, inBourne, G. H. ed.: The Biochemistry and physiology of Bone, New York,Academic Press, 1976: 1-59). There are a number of interrelationshipsbetween HA and bone formation:

1. HA is a prominent component of the extracellular matrix duringmorphogenesis of bone (B. Toole et al., Develop. Biol., 1971: 26, 28; H.Iwata et al., Clin. Orthop., Rel. Res., 1973: 90, 236);

2. Considerable quantities of HA are present during the transition ofmesenchymal cells to cartilage (O. Wiebkin et al., FEBS Lett., 1973: 37,42; C. H. Handley et al., Biochem. Biophys. Acta, 1976: 444, 69);

3. In terms of its correlation with wound healing processes and bonytissue development (B. P. Toole (1976) in S. H. Barondes, Ed., NeuronalRecognition, Plenum Press, New York, pp. 275-329), HA can be consideredas a sort of "primer".

It has long been known that HA is useful in the treatment of periodontaldiseases (Minerva Stomatol. 17: 140, 1968; Riv. Ital., Stomatolog. 20:1540, 1965). There seems to be well-defined sequence of events in thesecases:

--First, a HA-rich matrix is deposited in a space poorly furnished withcells;

--Secondly, cell migration is stimulated and the HA matrix isinfiltrated by cells migrating from adjacent tissues; and

--Lastly, the cells inside the extracellular matrix secretehyaluronidase (which degrades the HA), sulfated glycosaminiglycans, andcollagen, which replace part of the HA while the matrix becomesremodelled.

In each of the three developmental systems noted above, the HA matrix isfirst synthesized and then degraded. Consequently, it is probable thatthis transitory HA matrix, poor in cells, is needed before thecomplicated series of cell-mediated events which follows its destructioncan proceed.

The presence of a certain number of sites binding HA onto the differenttypes of cell surfaces has also been reported, such as in endothelialcells and fibroblasts (S. Eriksson et al., Exp. Cell Res. 1983: 144,223; R. H. Raja et al., J. Cell. Biol.; 1985: 101, 426a; C. B. Underhillet al., Cell Biol. 1979: 82, 475). Thus, HA is able to interactspecifically both with the cell surfaces and with the molecules of theextracellular matrices.

The chemical and physical-chemical characteristics involved in HA'sosteoinductive activity have not yet been identified. It is known thatthe biological activity of hyaluronic acid can often be associated withdefinite fractions of different molecular weights and viscosity.

SUMMARY OF THE INVENTION

Therefore, an object of the present invention is to provide differentfractions of hyaluronic acid extracted and purified from a source otherthan that of the cells on which it is to exert osteoinductive activity.Starting from the raw material, it is possible to obtain a substantiallypure fraction of hyaluronic acid with a molecular weight in the rangefrom about 20 to about 60 Kdaltons, a viscosity in the range from about1.2 dl/g to about 2.8 dl/g, and a protein content of less than about0.5% (weight/weight).

Another object of the present invention is to provide a pharmaceuticalcomposition, comprising a hyaluronic acid fraction possessingosteoinductive activity, and a pharmaceutically acceptable carrier. Saidpharmaceutical composition can be in the form of microspheres,membranes, films, non-woven tissues, tubes, nerve channels, sponges, apowder, or granules, and can contain from 0.1% to 99% by weight of saidhyaluronic acid fraction, more preferably from 10% to 90% by weight ofsaid hyaluronic acid fraction, and most preferably from 20% to 80% byweight of said hyaluronic acid fraction.

Further objects of the present invention include providing methods,useful in human and veterinary medicine, of maintaining bone function,of preventing the loss of bone function, of recovering bone functionunder traumatic conditions, of recovering bone function in acute orchronic pathological conditions, including acute pathological conditionsin a late stage and chronic degenerative pathological conditions, and oftreating pathological conditions caused by bone aging. These methodscomprise administering to a human or animal in need thereof an effectiveamount of a pharmaceutical composition comprising a hyaluronic acidfraction possessing osteoinductive activity and a pharmaceuticallyacceptable carrier.

Said pharmaceutical composition can be administered by a route selectedfrom the group consisting of parenteral administration, intradermaladministration, local administration, and topical administration. Localadministration can include intragingival application at the site of bonedefects.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will be better understood from the following detaileddescriptions taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention, in which:

FIG. 1 shows various methods employed to produce the low molecularweight fractions of hyaluronic acid described herein.

FIG. 2 shows the osteoinductive activity in vitro, as measured in termsof bone colony area, of hyaluronic acid fractions having molecularweights of 30 and 40 Kdaltons at concentrations of 0.5, 1.0, and 2.0mg/ml.

FIG. 3 shows the osteoinductive activity in vitro, as measured in termsof bone colony number, of hyaluronic acid fractions having molecularweights of 30 and 40 Kdaltons at concentrations of 0.5, 1.0, and 2.0mg/ml.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is provided to aid those skilled inthe art in practicing the present invention. Even so, the followingdetailed description should not be construed to unduly limit the presentinvention, as modifications and variations in the embodiments hereindiscussed may be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

The contents of each of the references cited herein are incorporated byreference in their entirety.

MATERIALS AND METHODS

Preparation of Low Molecular Weight Hyaluronic Acid Fractions fromHyalastine

EXAMPLE 1

Method for obtaining a mixture of HYALASTINE and HYALECTIN fractionswithout inflammatory activity

Hen crests, either fresh or frozen (3000 g), are minced in a meat mincerand then carefully homogenized in a mechanical homogenizer. Theresulting paste is placed in an AISI 316 stainless steel container or ina glass container with 10 volumes of anhydrous acetone. The entirecontent is then agitated for 6 hours at a speed of 50 g/minute and leftto separate for 12 hours, after which the acetone is syphoned off anddiscarded.

This extraction process is repeated until the discarded acetone hasreached the correct humidity level (Karl-Fischer method).

The resulting substance is then centrifuged and vacuum dried at asuitable temperature for 5-8 hours. With this process, approximately500-600 g of dry powder is obtained from the hen crests.

300 g of the dry powder is then submitted to an enzymatic digestionprocess with papain (0.2 g) through a buffered aqueous medium with aphosphate buffer in the presence of a suitable quantity of cysteinehydrochloride. This mixture is then agitated for 24 hours at 60 g/minuteat a constant temperature of 60°-65° C. This whole mass is cooled to 25°C. adding 60 g of Celite (R) and the agitation is maintained for anadditional hour.

The resulting mixture is filtered until a clear liquid is obtained. Thisclear liquid then undergoes molecular ultrafiltration by means ofmembranes with a molecular exclusion limit of 30,000 to retain on themembrane those molecules with a molecular weight of greater than 30,000.Five to six original volumes are ultrafiltered and, at the same time,distilled water is continually added to the product. The addition ofdistilled water is suspended and the product is ultrafiltered until itis reduced to one-third of its original volume.

The residue liquid is rendered 0.1M by adding sodium chloride and thetemperature is brought to 50° C. 45 g of cetylpyridinium chloride isadded while the product is being agitated at 60 g/minute. This mixtureis agitated for 60 minutes, after which 50 g of Celite(R) is added.Under agitation the temperature of the product is reduced to 25° C. andthe precipitate formed is collected by means of centrifugation. Theprecipitate thus obtained is suspended in a 0.01M solution of sodiumchloride (5 liters) containing 0.05% cetylpyridinium chloride. It isagitated for a further 60 minutes at 50° C. The temperature is loweredto 25° C. and the precipitate centrifuged.

The washing process is then repeated three times and the precipitatefinally gathered into containers holding 3 liters of a 0.05M solution ofsodium chloride containing 0.05% of cetylpyridinium chloride. This isagitated at 60 g/minute for 60 minutes and maintained at a constanttemperature of 25° C. for a period of 2 hours. The lipid supernatant iseliminated by means of centrifugation.

The procedure is thus repeated several times with a 0.1M sodium chloridesolution containing 0.05% of cetylpyridinium chloride. The mixture iscentrifuged and the supernatant discarded. The precipitate is dispersedin a 0.30M sodium chloride solution containing 0.05% ofcetylpyridiniumchloride (3 l). The mixture is agitated and both theprecipitate and the clear liquid are gathered. Extraction is repeated onthe precipitate an additional 3 times, each time using 0.5 liters of thesame aqueous solution.

Finally, the residue precipitate is eliminated and the clear liquidsunited in a single container. The temperature of the liquid is increasedto 50° C. while agitating. The liquid is then brought to 0.23M withsodium chloride. 1 g of cetylpyridinium chloride is added and agitationmaintained for 12 hours. The mixture is cooled to 25° C. then filtered,first through Celite(R) packs and then through a filter (1 μ).

The resultant mixture then undergoes a further molecular ultrafiltrationthrough membranes with a molecular exclusion limit of 30,000,ultrafiltering 3 original volumes with the addition of a 0.33M sodiumchloride solution. The addition of the sodium chloride solution issuspended and the volume of the liquid reduced to a quarter of itsoriginal volume.

The solution thus concentrated is precipitated under agitation (60g/minute) at a temperature of 25° C. with three volumes of ethanol(95%). The precipitate is collected by centrifugation and thesupernatant discarded. The precipitate is dissolved in 1 liter of 0.1Msodium chloride solution and the precipitation procedure is repeatedwith three volumes of 95% ethanol.

The precipitate is gathered and washed, first with 75% ethanol (threetimes), then with absolute ethanol (three times) and thirdly withabsolute acetone (three times).

The product thus obtained (HYALASTINE+HYALECTIN fraction) has an averagemolecular weight between 250,000 and 350,000.

The hyaluronic acid yield is equal to 0.6% of the original fresh tissue.

EXAMPLE 2

Method for obtaining the HYALASTINE fraction from the mixture obtainedby the method described in Example 1

The mixture obtained by the method described in Example 1 is dissolvedin pyrogen free distilled water in proportions of 10 mg of product in 1ml of water. The solution thus obtained undergoes molecularultrafiltration through membranes with a molecular exclusion limit of200,000 with a concentration technique and without addition of water onthe membrane. During the ultrafiltration process through membranes withan exclusion limit of 200,000, molecules with molecule weight greaterthan 200,000 will not pass, whereas smaller molecules will pass throughthe membrane along with the water; During the filtration process nowater is added in the compartment above the membrane; therefore, thevolume in this compartment will decrease, along with an increase in theconcentration of molecules with M.W. over 200,000. It is thenultrafiltered until the volume on the membrane is reduced to 10% of theinitial volume. Two volumes of pyrogen free bidistilled water are addedand the solution is again ultrafiltered until the volume is reduced toone-third. The operation is repeated another two times.

The solution which passes through the membrane is brought to 1.0M withsodium chloride and then precipitated with four volumes of ethanol at95%. The precipitate is washed three times with 75% ethanol and thenvacuum dried.

The product thus obtained (HYALASTINE fraction) has an average molecularweight between 50,000 and 100,000.

The hyaluronic acid yield is equal to 0.4% of the original fresh tissue.

The low molecular weight hyaluronic acid fractions of the presentinvention were prepared from the hyalastine fraction as shown in FIG. 1.

Method for the cultivation of isolated mesenchymal cells

Swiss Webster mice at their 12th-13th day of pregnancy were anesthetizedwith 0.4 ml of sodium nembutal administered intraperitoneally. Thefetuses were removed under sterile conditions. The epithelium wasremoved from the head, and the crown lifted to reveal the brain.Portions of the temporal, frontal, parietal and occipital mesenchymewere removed and immediately placed in BGJb medium. The tissue from eachmouse was placed separately in a sterile Petri dish containing 12 ml ofsterile Hank's balanced saline solution (HBSS) without calcium ormagnesium, 10 mg of raw collagenase, and 50 μg of dextrose. The Petridish was placed on a rotating platform and the tissue dissociated bycrushing for 90 minutes at 37° C. The tissue thus dispersed wastransferred to a sterile, conical 50 ml test tube for centrifugation.After centrifugation, the supernatant was removed and the resultingpellet was resuspended in BGJb medium enriched with 50 μg/ml gentamycinand 10% heat-inactivated fetal calf serum (checked for the absence ofviruses). This procedure is repeated three times to ensure completeremoval of collagenase. After the final resuspension, an aliguot istransferred to a hemocytometer for the determination of cell numbers. Ifthe values obtained from the two chambers of the hemocytometer differ bymore than the square root of the mean from the two chambers, the countis repeated. The cell suspension is then diluted or concentrated until aconcentration of 4×10⁵ cells/ml is obtained. Aliquots of 5 ml, eachcontaining 2×10⁶ cells, are placed in sterile, 60-mm Petri dishes andstored at 37° C., 100% humidity, in an atmosphere of 95% compressed airand 5% carbon dioxide. These cells develop into numerous bone colonies,as described by Marvaso and Bernard (J. S. Ko et al., Am. J. Anat. 1981:161, 415).

Microscopy

The bone cultures are fixed in situ and subsequently removed intact fromthe Petri dishes with a rubber scoop, and gently packed by low-speedcentrifugation. By sectioning the cell cultures thus treated, it ispossible to see many areas of the culture, including their reciprocalrelationships, and therefore to obtain an overall impression of thehistological representation in each Petri dish. After fixing, all thetissue is dehydrated by passage through a series of alcohols, clarifiedwith xylene, and embedded in paraffin. The area covered by each bonecolony is measured by a digital analyzer connected to a phase contrastmicroscope (35×). Sections of 5-6 microns are taken from along theentire length of the material and mounted in a semiserial manner. Threeconsecutive sections of each row of 15 are stained in the same orderwith toluidine blue, H. and E, or neutral red, having been treated withVon Kossa reagents for the determination of bound calcium. All sectionsof a given tissue are examined, and the most representative microscopicfields are photographed.

The control skulls, the isolated mesenchymal cell pellets, and the cellculture material are collected and fixed as described above, with thedifference that the cell cultures are not removed from the Petri dishes,but are fixed in situ. All the tissues are then post-fixed for one hourin 1% osmium tetroxide in 0.1M sodium cacodylate at pH 7.4, dehydratedin alcohol and propylene oxide or hydroxypropylmethacrylate and embeddedin Epon 812. The blocks are cut with glass blades on a Porter-blum MT-1ultramicrotome. For optical microscopy, semi-thin 1 micron sections ofplastic material are stained with aqueous toluidine blue, while otherthin sections are stained with uranyl acetate and lead citrate and usedfor all the ultrastructural analyses. Electron microscopy was performedwith a Siemens 1A Elmskop at 80 KV. Within the cell cultures, only theclumps which represent cell aggregates of bone colony are examined. Atleast three clumps are chosen at random from each Petri dish, embeddedat the set times.

EXAMPLE 3

Different quantities of Ha with different molecular weights were addedto the cultured bone colonies, prepared as previously described.

The samples of HA, in powdered form, represented different molecularweight fractions of hyaluronic acid: 2×10⁴ ; 4×10⁴ ; 6×10⁴ ; 16×10⁴ ;55×10⁴ ; 88×10⁴ ; and 13×10⁵.

The characteristics of the HA samples, identified as A thru G, are shownin Table 1

                  TABLE 1                                                         ______________________________________                                        Sample Mean molecular Intrinsic   % albumin                                   Code   weight (d × 10.sup.-3)                                                                 Viscosity dl/g                                                                            proteins                                    ______________________________________                                        A       20            1.20        <0.5                                        B       40            1.3         <0.5                                        C       60            2.8         0.3                                         D      160            4.8         0.4                                         E      550            12.1        0.2                                         F      800            10.0        0.2                                         G      1300           21.0        0.1                                         ______________________________________                                    

The various HA samples were used to treat mesenchymal cells at threedifferent concentrations: 0.5, 1.0 and 2.0 mg/ml. All samples wereadministered to a total of 8 Petri dishes per test to yieldstatistically significant data. The culture medium (BGJb) containing HAwas first replaced with HA-free medium after 48 hours, and then every 24hours thereafter until day 10, when the resulting tissues were fixed.

The photomicroscopic results showed the greatest osteoinductiveactivities in samples A, B and C.

FIGS. 2 and 3 show the effects of HA fractions having molecular weightsof 30 and 40 Kdaltons on colony area and colony number, respectively, incultured bone colonies.

As shown in FIG. 2, the HA fraction with a molecular weight of 30Kdaltons (left panel) was most effective in stimulating bone colony areadevelopment at a concentration of 1 mg/ml. The HA fraction with amolecular weight of 40 Kdaltons was most effective at a concentration of2 mg/ml (right panel). In both cases, the relationship between colonysize and HA dose was roughly linear.

As shown in FIG. 3, both the 30 and 40 Kdalton HA fractions were mosteffective in stimulating colony formation at 2 mg/ml. Treatment withboth HA fractions induced a marked increase in the number of bonecolonies in culture as compared to non-treated controls. This increasewas roughly linear with increasing dose, and was more evident with the30 Kdalton HA fraction.

The invention being thus described, it is obvious that the same can bemodified in various ways. Such modifications are not to be considereddivergences from the spirit and scope of the present invention, and anymodification that would be apparent to one skilled in the art is to beconsidered as coming within the scope of the following claims.

We claim:
 1. A method of stimulating growth of bone forming cellscomprising exposing mesenchymal cells to hyaluronic acid having amolecular weight of 20-60 kD.
 2. The method of claim 1, wherein saidhyaluronic acid has a viscosity in the range of from 1.2 dl/g to 2.8dl/g.
 3. The method of claim 1, wherein said hyaluronic acid has aprotein content of less than 0.5%.